This is a competitive revision application submitted in response to NOT-OD-09-058 entitled "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications". The parent grant is R01GM076360 entitled "Studies of naturally occurring structurally modified carbohydrates". Sialylated carbohydrate structures presented on the cell surface play diverse and important biological roles in cell-cell interaction processes. Our parental grant is focusing on the studies of carbohydrate-protein interactions using naturally occurring sialosides synthesized in our lab. In our preliminary studies, we have established efficient approaches for studying two types of sialic acid (Sia)-recognizing protein systems: bacterial sialidases and mammalian Siglecs. For sialidase (sialic acid-releasing exoglycosidase) studies, an efficient microtiter-plate-based high-throughput sialidase substrate specificity assay has been developed using para-nitrophenyl sialyldisaccharides obtained by an effective one-pot three-enzyme chemoenzymatic synthetic approach as substrates. For mammalian Siglecs studies, we have developed a combinatorial chemoenzymatic approach to obtain biotinylated sialosides which can be combined with microtiter-plate-based high-throughput format to find the preferred ligands for Siglecs without the tedious product purification process. Excited by the results obtained, we plan to take the advantages of the sialoside products produced as well as the chemoenzymatic synthetic strategies and the assay methods developed in the parent grant to expand the carbohydrate-protein interaction study to the detection of the preferred glycan ligands and potent glycan-based inhibitors for hemagglutinins on the surface of influenza A virus. In addition, substrate specificity of neuraminidases on the surface of different influenza A virus strains will be characterized and compared to that of human neuraminidases. We hypothesize that 1) the influenza virus NA substrate specificity is different among different viral strains, and is different from human and bacterial sialidases;2) a correlation exists between the viral HA ligand specificity and the viral NA substrate specificity. To test these hypotheses, two specific aims are to 1) determine the substrate specificity of recombinant human sialidases and neuraminidases on the surface of purified avian and human influenza A virus particles and 2) determine the ligand specificity and identify potential inhibitors of hemagglutinins on influenza A virus particles. We believe the completion of the proposed study will lead to 1) a better understanding of the relationship of ligand specificity of hemaglutinin and substrate specificity of neuraminidase of influenza viruses;2) finding a set of compounds that can be used as diagnostic tools for influenza virus infection;and 3) identifying potential inhibitors against viral hemagglutinin binding to host. PUBLIC HEALTH RELEVANCE: Influenza A virus is an acute viral disease of the respiratory tract that affects millions of people each year. The virus has two types of surface glycoproteins: hemagglutinin and neuraminidase. Both of these glycoproteins recognize sialic acid-containing structures on the host cell surface and play important roles in influenza virus infections. Hemagglutinin binds to the sialic acid- containing glycans on host respiratory cell surface and initiate infection. Neuraminidase is believed to cleave the sialic acid from the glycans on the surface of infected cells to release the newly formed viral particles to allow the spreading of virus. The studies proposed here can help to understand the relationship of the ligand specificity of hemagglutinin and the substrate specificity of neuraminidases and their involvement in infection. Molecules that can be used for diagnosis of influenza A virus infection and potential inhibitors that can against the binding and the infection of influenza virus can also be identified.